Genetically modified animals offer the potential for tremendous advances in the sustainable production of valuable pharmaceutical products, such as antibodies. However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species. The ability to incorporate genetic modifications encoding proteins into the DNA of a species for a specific expression requires several distinct technologies that must be developed for each genetic modification. One approach to alter the genetic and physical characteristics of an animal is to introduce cells into recipient embryos of the animal. These cells have the ability to contribute to the tissue of an animal born from the recipient embryo and to contribute to the genome of the offspring of a resulting genetically modified animal.
Significant expenditure of time and resources has been committed to the study and development of cell lines, the manipulation of the genome of the cells, and cell culture techniques that permit such engineered cells to be maintained in culture. Although many attempts have been made, the ability to sustain the pluripotency of engineered cells in culture has been achieved for only a few species, notably mice.
If sustainable cultures were readily available and susceptible to genetic engineering while maintaining pluripotency, a broad application of new technologies would be available. Because cultured cells can contribute to the tissues of a chimeric animal, the physiological characteristics of the animal from which an embryonic stem cell was derived can be transferred to a recipient embryo by incorporating these cells into the recipient animal in an embryonic state. This offers two principal advantages: first, the phenotype of an animal from which embryonic stem cells are derived can be selectively transferred to a recipient embryo. Second, as noted above, when the cell cultures are particularly stable, the genome of the cells can be modified genetically to introduce genetic modifications into a recipient embryo in which the cells are introduced.
In certain cases, the cells can be engineered with a transgene that encodes an antibody. The transgene is a genetic construct that contains DNA that acts as the blueprint for the production of the antibody and contains sufficient coding and regulatory elements to enable the expression of the antibody in the tissue of the animal that is created from the insertion of the cells into a recipient embryo. However, the collection of a valuable antibody from the tissues of an animal typically requires that the expression be limited to certain specific tissue types that facilitate collection of the expressed protein and convey desirable chemical properties. For example, in cows, the expression of a protein in the milk enables the ready collection of the protein by simply collecting the milk of the cow and separating the exogenous protein. In chickens, the robust production of antibodies in the white of the egg also provides an attractive vehicle for the expression and collection of the antibodies. Furthermore, where the tissue specific expression is specific to the oviduct of a chicken, the expression yields antibodies having certain specific desirable chemical properties that increase the therapeutic utility of the antibodies when used in the treatment of a human patient. Thus, one particularly attractive field of research and commercial development is genetically engineered chickens that selectively express antibodies in the egg to facilitate isolation and collection of proteins with desirable chemical properties. The ability to selectively produce exogenous antibodies in specifically selected cells of an animal is particularly valuable because the absence of tissue specificity simply results in the antibody being expressed in all of the tissues of an animal. Under such circumstances, it is unlikely that a meaningful quantity of the antibody could be separated from the animal, the ubiquitous expression of an exogenous antibody is usually very damaging to the overall health and well being of the animal, and the desirable chemical properties exhibited in the chicken oviduct are not present.
If cell culture is sufficiently stable to allow a transgene to become integrated into the genome of the cell, a transgene encoding tissue specific expression of an antibody can be passed to a new chimeric or transgenic organism by several different techniques depending on the target cell and the specific construct used as the transgene. Whole genomes can be transferred by cell hybridization, intact chromosomes by microcells, subchromosomal segments by chromosome mediated gene transfer, and DNA fragments in the kilobase range by DNA mediated gene transfer (Klobutcher, L. A. and F. H. Ruddle, Annu. Rev. Biochem., 50: 533-554, 1981). Intact chromosomes may be transferred by microcell-mediated chromosome transfer (MMCT) (Fournier, R. E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A., 74: 319-323, 1977). The specific design of the transgene also must consider the content of the DNA sequences encoding the antibody, the target cell line, the specific tissue in which expression is targeted, the host organism in which expression occurs, and the antibody to be expressed. The transgene designed for tissue specific expression must satisfy several parameters to enable successful integration into the genome of a cell and to insure successful expression in the selected tissue of the host organism.
As noted above, the introduction of genetic modifications to produce transgenic animals has been demonstrated in only a very few species. For mice, the separate use of homologous recombination followed by chromosome transfer to embryonic stem (ES) cells for the production of chimeric and transgenic offspring is well known. Powerful techniques of site-specific homologous recombination or gene targeting have been developed (see Thomas, K. R. and M. R. Capecchi, Cell 51: 503-512, 1987; review by Waldman, A. S., Crit. Rev. Oncol. Hematol. 12: 49-64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4: 761-763, 1994), and manipulation and selection of chromosome fragments by the Cre-loxP system techniques (see Smith, A. J. et al., Nat. Genet. 9: 376-385, 1995; Ramirez-Solis, R. et al., Nature 378: 720-724, 1995; U.S. Pat. Nos. 4,959,317; 6,130,364; 6,091,001; 5,985,614) are available for the manipulation and transfer of genes into murine ES cells to produce stable genetic chimeras. Many such techniques that have proved useful in mammalian systems would be available to be applied to non-mammalian systems if the necessary long term cell cultures were available and if transgenes could be designed that yielded tissue specific expression in specific tissues that facilitate isolation and collection of the exogenous protein.
The transgenes that enable tissue specific expression are complex and the genetic manipulations that are necessary to incorporate the transgenes into a recipient cell line require extensive manipulation that can threaten the pluripotency of the cells unless the culture conditions are optimized. Thus, cell lines suitable for use in transgenesis must be both stable in culture and must maintain pluripotency when the cell is transfected with a genetic construct that is large and complex enough to contain all of the elements necessary for tissue specific expression. In the resulting animal, the transgene must be effectively expressed in specific individual tissue types in which the transgene is designed to be expressed, and should not be expressed in other tissues such that the viability of the animal or the advantageous chemistry of the resulting protein is compromised.
For the production of exogenous antibodies, avian biological systems offer many advantages including efficient farm cultivation, rapid growth, and economical production. Further, the avian egg offers an ideal biological design, both for massive synthesis of antibodies and ease of isolation and collection of product. Furthermore, as described below in the context of the present invention, advantages of the chicken-based expression system compared to vertebrate, plant, or bacterial cell systems can be obtained for large quantities of antibody product.